Technical Field
The present invention is related to an agarase, especially to an agarase produced via a prokaryotic cell expression system.
Description of Related Art
Agar is a hydrophilic polysaccharide extracted from cell walls of red algae such as Gelidium spp., Gracilaria spp., Porphyra spp., and etc, and the main components thereof are agarose and agaropectin. Agarose is a neutral polysaccharide with α-1,3 and β-1,4 glycosidic linkage, which is capable of forming gel and has a molecular weight of at least 100 kDa. Agaropectin is not capable of forming gel and has a molecular weight of at most 20 kDa. Agaropectin has a similar composition with agarose while some hydroxyl groups of 3,6-anhydro-α-L-galactose thereof are replaced with methoxy, sulfoxy or pyruvate groups.
Hydrolase capable of hydrolyzing agar is named agarase, which can be classified as α-agarase (EC 3.2.1.158) and β-agarase (EC 3.2.1.81) in accordance with the hydrolysis site thereof. α-Agarase hydrolyzes at the α-1,3 glycosidic linkage of agarose and agaropectin and results in agaro-oligosaccharides having 3,6-anhydro-α-L-galactose group at the reducing end thereof. β-Agarase hydrolyzes at the β-1,4 glycosidic linkage of agarose and agaropectin and results in neoagaro-oligosaccharides having D-galactose group at the reducing end thereof.
There are many applications for agarase. Case in point, agarase can be used in molecular biology research for recovery of DNA from agarose Gel, can be used in cartilage tissue engineering as agar substrate for supporting cartilage cells and thereby facilitating cartilage cells purification, increasing collagen content, and improving the culture of cartilage tissue; can be used for preparing agaro-oligosaccharide and neoagaro-oligosaccharide; can be used for preparing algae protoplast for DNA transformation and cell fusion; can be used for hydrolysis of algae polysaccharides and speculating the structure of the algae polysaccharides based on the hydrolysis product; can be used for preparing algae single cell being used as feed of marine animal breeding.
Furthermore, the current researches have proved the oligosaccharides obtained by hydrolyzing agar or algae polysaccharide crude extract exhibit several physiological and biological activities, such as antioxidation, immune regulation, antibacterial, tyrosinase suppression, moisturizing, being used as prebiotic, decreasing serum total cholesterol, and etc. The oligosaccharides can also be the new generation of high value functional oligosaccharides, which are widely applied in cosmetic, health food, and pharmaceutical industries. There are several microorganisms being proved to be able to produce agarase; nevertheless, the production of agarase by those known microorganisms encounters lost of difficulties and defects unfavorable for mass production in the industries, for instance, insufficient production, unstable production, safety concern to the bacterial used, high production cost and etc.
In light of the foregoing, the researchers in the field have considered using acid hydrolysis method to hydrolyze agar or algae polysaccharide crude extract to obtain the required oligosaccharide. However, although conventional acid hydrolysis method is able to obtain agaro-oligosaccharide mixtures, it is unable to obtain products having uniform degree of polymerization. In comparison with acid hydrolysis method, enzymatic hydrolysis has several strengths and thereby is more ideal than acid hydrolysis method. The strengths includes enzymatic selectivity in cutting specific types of glycosidic linkages to obtain oligosaccharides of desired polymerization, ease in controlling degradation condition, temperature required for enzymatic reaction is lower than that of acid hydrolysis method therefore the energy consumption is decreased, ease in operation comparing with acid hydrolysis method wherein processes like acid-base neutralization and desalination are not required, chemical agents are not necessary therefore the operation is safer and less possible in contaminating environment, and agaro-oligosaccharide and neoagaro-oligosaccharide can be obtained.
To sum up, in order to facilitate the industrial applications of the oligosaccharides obtained from agarase hydrolysis of agar or algae polysaccharide crude extract, there is a need of novel agarase to provide more options for the field. Moreover, there is also a need of a production method of agarase, which can be operated in lower cost so that the production cost of the aforesaid oligosaccharides can be decreased for facilitating commercialization.